PROJECT SUMMARY Allogeneic hematopoietic stem cell transplantation (HSCT) involves the transfer of healthy donor hematopoietic cells, including hematopoietic stem cells and mature immune effector cells, to recipients with high-risk hematologic malignancies. The success of HSCT is fundamentally dependent on engraftment of normal donor- derived hematopoiesis. Inadequate graft function can cause a range of complications that impact recipient outcomes, including disease relapse, graft versus host disease, and infection. In preliminary studies, we identified healthy stem cell donors with clonally restricted hematopoiesis, marked by mutations in canonical genetic drivers of myeloid malignancies, where the aberrant clone engrafted in a transplant recipient, underwent selective expansion, and was associated with abnormal hematopoietic function. While rare patients developed donor cell leukemia after long latency, our data suggest that non-malignant outcomes of donor-engrafted clonal hematopoiesis, such as hematopoietic dysfunction or graft versus host disease may be more common and may manifest earlier after transplantation, thereby contributing significantly to transplant-related morbidity. We hypothesize that the presence of clonal hematopoiesis of indeterminate potential (CHIP) is an age-independent predictor of donor hematopoietic fitness that negatively impacts recipient outcome by causing impaired graft function. This proposal combines complementary genetic, functional, and transcriptomic approaches in a large cohort of stem cell donor-recipient pairs to define the impact of donor CHIP on allo HSCT outcomes. In Specific Aim 1, we will determine the frequency and clinical significance of CHIP in a 1911 allogeneic stem cell donors 40 years of age and older (discovery cohort,n=1189; external validation cohort, n=722). To complete this aim, we have developed and validated a highly sensitive sequencing platform for identification of CHIP in donor samples, with >50-fold greater sensitivity than standard next generation sequencing modalities. This work will be closely linked to Specific Aim 2, where we will focus on the subset of donors with clonal mutations to define the efficiency and lineage potential of clonal stem cell engraftment, and the genetic evolution of clones over time. Finally, in Specific Aim 3 we will dissect the functional impact of donor CHIP on immune function in transplant recipients, testing the hypothesis that stem cell clones can perturb inflammatory cytokine production and proper recovery of immune activity via their clonal contribution to mature immune cell subsets. Together, the proposed studies may define a new paradigm of donor-attributable risk in allogeneic HSCT and provide insights into biological mechanisms of clonal dominance and the influence of microenvironmental context on clonal evolution.